Long-range charge transfer mechanism of the III2IV2 mycobacterial supercomplex

Aerobic life is powered by membrane-bound redox enzymes that shuttle electrons to oxygen and transfer protons across a biological membrane. Structural studies suggest that these energy-transducing enzymes operate as higher-order supercomplexes, but their functional role remains poorly understood and highly debated. Here we resolve the functional dynamics of the 0.7 MDa III2IV2 obligate supercomplex from Mycobacterium smegmatis, a close relative of M. tuberculosis, the causative agent of tuberculosis. By combining computational, biochemical, and high-resolution (2.3 Å) cryo-electron microscopy experiments, we show how the mycobacterial supercomplex catalyses long-range charge transport from its menaquinol oxidation site to the binuclear active site for oxygen reduction. Our data reveal proton and electron pathways responsible for the charge transfer reactions, mechanistic principles of the quinone catalysis, and how unique molecular adaptations, water molecules, and lipid interactions enable the proton-coupled electron transfer (PCET) reactions. Our combined findings provide a mechanistic blueprint of mycobacterial supercomplexes and a basis for developing drugs against pathogenic bacteria.


Content
Extended discussion: Functional relevance of mycobacterial adaptions at the Qo site                 Table S1.List of atomistic molecular dynamics simulations (ca. 10 µs).Table S2.List of subunits in the simulation models.Table S3.List of residues modelled in non-standard protonation states based on Monte Carlo electrostatic calculations.Table S4.Calculated electron transfer rates and estimated parameters.Table S5.Calculated average redox midpoint potentials of cofactors based on MD simulations.Table S6.Cryo-EM data collection, refinement, and validation statistics.Table S7.Atom types and partial charges for MQ cofactors.Table S8.Atom types and partial charges for the FeS centre.Table S9.Atom types and partial charges for heme b.Table S10.Atom types and partial charges for heme c.Table S11.Atom types and partial charges for Cu active site in SodC.Table S12.Validation of the menaquinone geometry.Table S13.Validation of the FeS geometry.Table S14.Validation of heme b geometry.Table S15.Calculated pKa values of selected residues in the Qo1a and Qi sites.

Extended discussion: Functional relevance of mycobacterial adaptions at the Qo site
To probe the functional relevance of the modular adaptations in the Qo1a site, we studied redox tuning effects by in silico mutagenesis and electrostatic models (see Methods).In the D309N variant, the Rieske redox potential is downshifted by ca. 100 mV and heme bL is upshifted by 50 mV relative to the WT model (Fig. 6f, Supplementary Table 5), whereas the menaquinone (MQ/SQ) has a predicted Em of around -150 mV, i.e., somewhat below its Em value of -80 mV in membranes.These findings suggest that Asp309, which is replaced by a tyrosine in the canonical Complex III (Supplementary Fig. 20), could favour the electron transfer and increase the thermodynamic driving force for the menaquinone oxidation process.Protonation of His355 (by PCET from QH2) increases the redox potential of the FeS Rieske centre by ca.190 mV (+90 mV→+280 mV), leading to an effective proton-coupled redox potential of +120 mV (Fig. 6f).This protonation is favoured by the Asp309, which has an important redox-tuning effect on the Qo1a site, and helps in establishing a downhill electron transfer between semiquinone and heme bL, a process that is more favoured with the ubiquinol (+90 mV).The D309N substitution leads to a significant decrease in the hydration of the proton pathway next to the Rieske FeS centre, indicating that Asp309 could also support the proton release (Supplementary Fig. 5c).
The electron transfer between the quinone and heme bL is linked to a proton transfer from the semiquinone (QH • ) to Asp302 (Fig. 5b, d), which is adjacent to His110.Protonation of this site favours the closed conformation of the ion-pair between His110 and Asp302 (Supplementary Figs.9e, 5d, e), whereas deprotonation of His110 leads to an ion-pair opening and increase of the hydration state of the proton pathway next to heme bL (Supplementary Fig. 5d, e).The His110/Asp302 ion-pair is resolved in a closed conformation in our cryoEM structures (Fig. 3f), resembling the conformation observed prior to deprotonation of the quinol in our MD simulations (see Supplementary Fig. 5e)        .Parts of the protein that are located in the membrane plane are coloured in blue.Waters are shown as red spheres and cofactors or cardiolipin as light green sticks.. b, Simulations (S1/S7, Supplementary Table 1) show rapid increase in buried water molecules during the first 50 ns and reach a plateau after ca.300 ns.c, d, Hydration levels in the putative proton exit pathways, leading from the Qo1a site to the P-side bulk.The D302N mutation results in a significant decrease in the hydration level at the Qo1a site.The hydration along the heme pathway depends on the interaction between Asp302 and His110 in the MD simulations, resulting in lower hydration levels in the closed ion-pair conformation (see panel e).e, The His110-Asp302 distance during MD simulations, show a stable salt-bridge when His110 is modelled in a protonated state, and a more dynamic hydrogen-bond when Asp302 or His110 are neutral.The D302N mutation and the neutral His110 state result in dissociation of the residues during the MD simulations.Raw hydration data is shown is shaded lines, while the smoothed data is shown in solid lines.c, d The Q in Qo1b forms π-stacking interactions with Phe153, as well as non-polar interactions with several other residues.e, f The Q in Q2 forms π-stacking interactions with Trp276, as well as with other non-polar residues.g, The edge-to-edge distance between Q and the FeS Rieske centre depend on the redox state of the quinone.The menaquinone binds in a stable binding pose in Qo1a, especially in all anionic charged states, although transient unbinding events (distance > 3.5 Å) can be observed.Protonation of His110 stabilises the quinone binding.h-j, Estimates of binding free energies (from PBSA-MM calculations) of QH2, QH • , and Q in the different sites calculated at ε=10.Similar affinities are observed for all three sites, but with a somewhat overall stronger binding in the Qo1a site relative to Qo1b / Qo2.k, Distance map showing the Q position relative to the FeS and heme bL cofactors for different trajectories.In one trajectory (dark blue), quinol is dynamically more flexible, and dissociates from Qo1a close to Qo1b.For each donor-acceptor pair, the packing densities and electron rates were calculated along all possible donor-acceptor pair, by considering the explicit intervening protein surrounding between the groups (see Methods).Left: Snapshot of the donor-acceptor groups with example distances (edgeto-edge, mean, and max distance).Right: Distribution of pairwise distances, packing densities (ρ) and calculated electron transfer rates (k).The average electron transfer rate, calculated as sum of the coupling-weighted rates, is marked with a red line.S1).The canonical Complex IV (CCO, bovine) exhibits fast water exchange with the bulk (t1/2=23 ns), while the supercomplex (SC) shows a significantly longer water exchange rate (t1/2=70 ns).c, Ion-pair distances between Asp115-Arg527, Asp115-Lys38, Asp36-Arg527 and Asp36-Lys38 at the D-channel entrance.The ion-pairs remain mostly closed, but transient opening conformations are observed.d, Comparison of the volumes of the D-and K-channels, estimated using CAVER3 10 .The D-channel has nearly three times the volume of the K-channel (134 Å 3 vs 47 Å 3 , respectively).e, Water survival probabilities in the D-and K-channel (see Methods) suggest that water molecules have a higher mobility in Kchannel, and could rationalise why less water is resolved in the K-channel in the experimental structures of Complex IV.Shaded lines show the individual channels, while the solid line represent the average between them.S12-S14.The MM and QM/MM optimisations were initiated from independent snapshots followed by a geometry optimisation.The MM system was minimised for 10000 steps, while the QM/MM system was minimised until energy convergence threshold of 0.006 kcal mol - 1 , with the MM region restrained.The data raw data for the box plots can be found in Supplementary Tables 12-14.

Figure S24. Schematic overview of charge transfer and quinone diffusion energetics.
Representation of the energetics of the local PCET reaction at the Q1a site, and the binding and release of the quinol/quinone from the membrane pool.Binding of quinol to the QH2,o1b and QH2,o1a sites from the membrane pool is exergonic by a few kcal mol -1 (based on Km∼120 μM (Ref. 2 ) and calculated binding affinities (see also Supplementary Fig. 8).The two local binding sites could provide entropic stabilisation into the binding process.The QM/MM calculations predict that the initial PCET is weakly exergonic, followed by an endergonic PCET towards heme bL.The green curve accounts for the subsequent electron transfer and proton translocation processes, as well as the quinone unbinding, including the cost for moving charges against the pmf (here 200 mV) based on experimental redox potentials 12 .The subsequent electron transfer and proton translocation processes are exergonic, whilst the whole process is driven by the reduction of O2 at the BNC (Em∼820 mV).
Table S1.List of atomistic molecular dynamics simulations (ca. 10 µs).All simulations were based on the complete system, with around 900,000 atoms modelled in the symmetric (sym) and asymmetric (asym) states (see Methods).The oxidised FeS Rieske centre was modelled in a Fe 3+ Fe 3+ state and deprotonated H355 (e-tautomer), and the reduced state with Fe 3+ Fe 2+ and both H335/H355 in their neutral form.Heme bH, heme c1, heme c2, CuA, and the Cu of SOD-C were modelled in their oxidised states, whereas the binuclear centre was simulated in the PM state (Fe IV =O 2-/Cu II -OH -/Tyr-O • ).Simulations S17+S18 are performed of the canonical bovine CcO (Bt CcO, see Ref. 12 ), and used as a reference for the D-channel hydration analysis (Fig. S10).Cox I His264 (HSE), Glu266, Asp385

Simulation
Cox II His232 (HSE), His281 (HSE) Table S4.Calculated electron transfer rates and estimated parameters.Rates in light grey were excluded from the kinetic model, since these reactions were modelled as irreversible to account for Qi unbinding and oxygen reduction.A detailed version of the kinetic model was created to account for proton-coupled modulation of the BNC redox potential.a generic value, cf.Ref. 13 .b from Ref. 14 c Computed from the MD trajectories, d from Ref. 3 .PLS -unprotonated proton loading site (prior to proton uptake via the D-channel); PLSH -protonated proton-loading site (after proton uptake from the D-channel).

Figure S1 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S2 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S6 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S7 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S8 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S9 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S10 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S11 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S12 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S14 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S15 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S16 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S17 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S18 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S19 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S20 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Figure S21 .
Figure S1.Details of the structural model of the SC.Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.Figure S4.SodC interaction and dynamics.Figure S5.Dynamics of buried water molecules in the SC.Figure S6.Comparison of MD and cryoEM water molecules.Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.Figure S8.Quinone dynamics in the Qo sites.Figure S9.Dynamics of the PDFY loop in the Qo1a site.Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.Figure S12.Details of the electron transfer rate calculations.Figure S13.Kinetic models of electron transfer in the supercomplex.Figure S14.QM/MM dynamics at the Qi site.Figure S15.CryoEM data analysis and validation of the 2.3 Å map.Figure S16.CryoEM data analysis and validation of the 2.8 Å map.Figure S17.Example cryoEM densities of lipid modelled based on the of the 2.3 Å map model.Figure S18.Hydration dynamics in the D-and K-channel.Figure S19.Computed redox potentials around the Qo1a site.Figure S20.Multiple sequence alignment of QcrB.Figure S21.Forcefield benchmarking and parameterisation.Figure S22.SC activity.Figure S23.Force field validation.Figure S24.Schematic overview of charge transfer and quinone diffusion energetics.

Movie S1 .
Molecular dynamics of the SC from M. smegmatis.Movie S2.Quinol oxidation-driven PCET to the FeS centre based on QM/MM free energy calculations.Movie S3.Quinol oxidation-driven PCET to the heme bL based on QM/MM free energy calculations.Movie S4.Protonation dynamics of the fully reduced Qi based on QM/MM MD simulations.Movie S5.Protonation dynamics of the semiquinone Qi based on QM/MM MD simulations.

Figure S1 .
Figure S1.Details of the structural model of the SC (based on PDB IDs: 6HWH 1 and 6ADQ 2 ).a, Symmetric model of the supercomplex showing modelled cardiolipins, as well as a SOD-C dimer (based on a homology model of PDB ID: 1PZS 3 ).b, Asymmetric model of the supercomplex featuring one open QcrC and the absence of LpqE.c, The SOD-C dimer, containing a Cu ion in its active site, and featuring an N-terminal lipid modification anchoring it to the membrane.d, The LpqE subunit with the same lipid anchor as the SOD-C.e, The QcrC subunit, comprising two c-type hemes, which bridge the electron transfer chain between Complexes III and IV in the closed conformation.The open, disconnected conformation arises due to the absence of LpqE.f, The AscF/G subunits, located in the vicinity of the entrance of the D-channel in Complex IV, with a possible regulatory role.g, PRSAF1 (prokaryotic respiratory supercomplex association factor 1) subunit is located at the interface of Complexes III and IV.h, Overlay of the canonical bc1 complex (in red colours; PDB ID: 1KYO 4 )with our current cryo-EM model (blue colours) aligned via the QcrB subunit (transparent).QcrC (cyan) occupies a similar position as cyt c1 (yellow), but has heme c1 in a different position, whereas LpqE (blue) partially overlaps with the bound cyt c (orange).The FeS cluster is shifted closer to the Qo site in the SC as compared to the canonical complex, where the Rieske protein is mobile.i, The quinol was modelled in the Qo1a site based on structural data of quinol and stigmatellin binding in canonical Complex III (PDB IDs: 6Q9E 5 , 1PP9 6 , c.f. Methods).The predicted binding site Qo1a in the supercomplex (in blue) in comparison to ubiquinone (in red) and stigmatellin (in green) from the canonical Complex III.

Figure S2 .
Figure S2.Molecular dynamics setup of the Mycobacterium smegmatis III2IV2 supercomplex.The protein is embedded in POPC lipid membrane and solvated in a waterbox and neutralised with 150 mM NaCl.The protein is shown in cartoon representation, the lipids as sticks, the ions as spheres, and water molecules as blue dots.The MD system comprises ca.930,000 atoms.See Tables S1-S3 for simulation setup details.

Figure
Figure S3.B-factors calculated from MD simulations and from cryoEM experiments.The simulations show an overall similar B-factor distribution in the different models.The dynamics of SOD-C was not included in this analysis to its high flexibility.LpqE and AscF/G show a high flexibility in the symmetric structure, while the asymmetric structure has an open QcrC conformation and is dynamically quite mobile.The membrane subunits show a high rigidity.See Ref. 1,7-9 for other cryoEM models.

Figure S4 .
Figure S4.SOD-C interaction and dynamics.a, SOD-C forms contacts with the QcrC subunit in simulation S13, by establishing ion-paired contacts between the subunits, in addition to unspecific non-polar contacts.b, Edge-to-edge distances between the SOD-C copper and heme c of QcrC (top: symmetric model, bottom: asymmetric model).Only the shortest distance is plotted.The distribution suggests that SOD-C does not form specific contacts with the supercomplex but sample many non-specific conformations, consistent with the blurred cryoEM densities.Shaded lines represent raw data while the solid lines show the smoothed data.c, SOD-C shows a blurred density map, consistent with the high mobility of the subunit in MD simulations.Density shown at a threshold of 0.22.

Figure S5 .
Figure S5.Dynamics of buried water molecules in the SC.a, Snapshot of buried water molecules in the membrane plane and in the vicinity of the cofactors (simulation S1, SupplementaryTable1). Parts of the protein that are located in the membrane plane are coloured in blue.Waters are shown as red spheres and cofactors or cardiolipin as light green sticks.. b, Simulations (S1/S7, Supplementary Table1) show rapid increase in buried water molecules during the first 50 ns and reach a plateau after ca.300 ns.c, d, Hydration levels in the putative proton exit pathways, leading from the Qo1a site to the P-side bulk.The D302N mutation results in a significant decrease in the hydration level at the Qo1a site.The hydration along the heme pathway depends on the interaction between Asp302 and His110 in the MD simulations, resulting in lower hydration levels in the closed ion-pair conformation (see panel e).e, The His110-Asp302 distance during MD simulations, show a stable salt-bridge when His110 is modelled in a protonated state, and a more dynamic hydrogen-bond when Asp302 or His110 are neutral.The D302N mutation and the neutral His110 state result in dissociation of the residues during the MD simulations.Raw hydration data is shown is shaded lines, while the smoothed data is shown in solid lines.

Figure S6 .
Figure S6.Comparison of MD and cryoEM water molecules.a, Comparison of experimentally resolved water molecules from cryoEM (in purple) with water molecules predicted by MD simulations (in red).The figure shows water molecules from the last snapshot of simulation S1 that are within 2 Å of an the experimentally identified water molecules.The MD simulations and cryoEM predict overall highly similar water positions.b, Proton pathways in Complex IV, leading from the proton loading site above heme a3 to the P-side of the membrane via the Mg 2+ ion.One output pathway leads to the P-side close to the CuA site, while the other pathway bridges D255 and K123 of subunit II to the P-side.

Figure S7 .
Figure S7.Example cryoEM densities and BNC models based on the 2.3 Å map.a, An AcPIM2 lipid is found next to the top of the QcrC helix, in close proximity of Lys197 and Asp209, suggesting its structural importance.b, The H-channel region of the supercomplex is conserved, but barely hydrated compared, as further corroborated by MD simulations, implying that the region does not conduct protons.c, Glu266 at the end of the D-channel, resolved in a downwards conformation.d, A Ca 2+ ion in Complex IV coordinated by Glu65 and Gln72, three backbone oxygens and two water molecules, modelled with 0.5 occupancy, is located 12 Å heme a. e-j, Geometry optimised QM models based on the cryoEM map, with different ligands modelled in the density between the Fe 3+ and Cu 2+ .

Figure S8 .
Figure S8.Quinone dynamics in the Qo sites.a-f, Edge-to-edge distance distributions between Q in the different Q sites and nearby residues.Only residues within 4 Å of Q in at least 20% of the MD trajectory are shown.The fraction of contacts is reported on the top left of the figure.a, b, The Q in Qo1a forms hydrogen-bonded contacts with His355 and Tyr159, as well as other non-specific polar and aromatic residues.c, d The Q in Qo1b forms π-stacking interactions with Phe153, as well as non-polar interactions with several other residues.e, f The Q in Q2 forms π-stacking interactions with Trp276, as well as with other non-polar residues.g, The edge-to-edge distance between Q and the FeS Rieske centre depend on the redox state of the quinone.The menaquinone binds in a stable binding pose in Qo1a, especially in all anionic charged states, although transient unbinding events (distance > 3.5 Å) can be observed.Protonation of His110 stabilises the quinone binding.h-j, Estimates of binding free energies (from PBSA-MM calculations) of QH2, QH • , and Q in the different sites calculated at ε=10.Similar affinities are observed for all three sites, but with a somewhat overall stronger binding in the Qo1a site relative to Qo1b / Qo2.k, Distance map showing the Q position relative to the FeS and heme bL cofactors for different trajectories.In one trajectory (dark blue), quinol is dynamically more flexible, and dissociates from Qo1a close to Qo1b.

Figure S9 .
Figure S9.Dynamics of the PDFY loop in the Qo1a site.Data from MD simulations S1 (a, QH2), S2 (b, QH2, protonated H110), and simulation S7 (c, oxidised Q).The PDFY loop is shown in purple.The Asp302 conformations are coloured by the simulation time (red to blue).d, Sequence conservation of the PDFY loop across different species.e, In simulations with a protonated His110 (simulations S2, S9, S12), the residue forms a stable ion-pair with Asp302, whereas Asp302 is dynamically highly flexible in the absence of the ion-pair (neutral His110, S1, S3, S5).

Figure S10 .
Figure S10.Example cryoEM densities of key regions based on the of the 2.8 Å map model.The figure shows water molecules around the Qo1a site; the menaquinone and cardiolipin molecules at the Qi site; and resolved water molecules around propionate region of heme a3.Waters are show as red spheres.

Figure S11 .
Figure S11.QM/MM free energy simulations of the PCET reactions in the Qo1a site.The QM/MM reaction coordinates were defined as a linear combination of bond-breaking and bond-forming distances for a, the first PCET from menaquinol to the Rieske FeS-centre and b, the second proton transfer from menaquinol to Asp302 coupled to electron transfer to heme bL.c, d, Convergence (top) and overlap (bottom) of the free energies for c, the first and d, the second PCET step.e, f, Spin densities for the reactant, transition, and product state of e, the first and f, the second PCET step.The densities are shown as red and blue volumes.g, Overview of the QM/MM simulation system.The location of the QM region is indicated by the red rectangle.

Figure S12 .
Figure S12.Details of the electron transfer rate calculations.For each donor-acceptor pair, the packing densities and electron rates were calculated along all possible donor-acceptor pair, by considering the explicit intervening protein surrounding between the groups (see Methods).Left: Snapshot of the donor-acceptor groups with example distances (edgeto-edge, mean, and max distance).Right: Distribution of pairwise distances, packing densities (ρ) and calculated electron transfer rates (k).The average electron transfer rate, calculated as sum of the coupling-weighted rates, is marked with a red line.

Figure S13 .
Figure S13.Kinetic models of electron transfer in the supercomplex.a, Edge-to-edge distance distributions from MD simulations S1, S3, S5, S6, and S7 (symmetric states).All predicted rates are within physiologically relevant timescales for the electron transfer.b, Kinetic model of the symmetric complex with the terminal electron transfer from Qo1a to heme bL, modelled as an irreversible step.c, Model of the electron transfer using Qo1a→Rieske and Qo1a→heme bL rates calculated from the QM/MM barriers based on transition state theory.d, Electron transfer in the closed QcrC protomer of the asymmetric supercomplex.e, Electron transfer kinetics for electron "leakage" from the open to the closed protomer, shows a much slower electron transfer rates as compared to the closed protomer.f, Electron "leakage" model accounting for the 4 ms proton uptake via the D-channel

Figure S15 .
Figure S15.CryoEM data analysis and validation of the 2.3 Å map.a, Example of micrograph showing particle orientation and sample homogeneity.b, Example of the 2D-class averages with views of different particle orientation.c, Fourier shell correlation (FSC) curve of the final 3D-reconstruction.d) Viewing direction distribution of particles.e, Estimation of the local resolution.f, Classification tree and particle sorting approach.

Figure S16 .
Figure S16.CryoEM data analysis and validation of the 2.8 Å map.a, Example of micrograph showing particle orientation and sample homogeneity.b, Example of the 2D-class averages with views of different particle orientation.c, Fourier shell correlation (FSC) curve of the final 3D-reconstruction.d) Viewing direction distribution of particles.e, Estimation of the local resolution.f, Classification tree and particle sorting approach.

Figure S18 .
Figure S18.Hydration dynamics in the D-and K-channel.a, Charged and polar residues at the entrance of the Dchannel.b, Survival probability of water molecules at the entrance of the D-channels (simulations S1, S7, S17+18, TableS1).The canonical Complex IV (CCO, bovine) exhibits fast water exchange with the bulk (t1/2=23 ns), while the supercomplex (SC) shows a significantly longer water exchange rate (t1/2=70 ns).c, Ion-pair distances between Asp115-Arg527, Asp115-Lys38, Asp36-Arg527 and Asp36-Lys38 at the D-channel entrance.The ion-pairs remain mostly closed, but transient opening conformations are observed.d, Comparison of the volumes of the D-and K-channels, estimated using CAVER310 .The D-channel has nearly three times the volume of the K-channel (134 Å 3 vs 47 Å 3 , respectively).e, Water survival probabilities in the D-and K-channel (see Methods) suggest that water molecules have a higher mobility in Kchannel, and could rationalise why less water is resolved in the K-channel in the experimental structures of Complex IV.Shaded lines show the individual channels, while the solid line represent the average between them.

Figure S19 .
Figure S19.Computed redox potentials around the Qo1a site.a-d, Calculated redox midpoint potentials from simulations of wild type SC (with and without protonated His110), as well as for respective in silico mutants (with His110 protonated).e, MD-averaged redox potentials relative to the menaquinone potentials and f, MD-averaged absolute redox potentials.

Figure S20 .
Figure S20.Multiple sequence alignment of QcrB.The analysis was performed using Clustal Omega 11 .

Figure S21 .
Figure S21.Forcefield benchmarking and parameterisation.a, b, The validity of the quinone parameters was benchmarked against DFT calculations (QM level: B3LYP-D3/def2-TZVP). MQ and His coordinates were independently optimised in vacuum and interaction energies were calculated at both QM and MM level at specific distances.QM interaction energies were also calculated with dielectric constants of 1, 4, and 80. c, Binding energies between MQ and the FeS centre resulted in similar interaction energies: (-11 kcal mol -1 QM model; -10 kcal mol -1 MM model).d-l, Cofactor models used for the force field parameterisations (see Methods for further details).

Figure S22 .
Figure S22.SC activity.Raw traces of the coupled activity measurements.The grey line shows the background O2 reduction in the absence of SC and was subtracted from the other activities.Shaded lines represent the raw data, while solid lines show smoothed data.

Figure S23 .
Figure S23.Force field validation.Comparison of differences in bond lengths and angles between MM (iceblue) and QM (orange) minimised structures of a, b) quinone, c, d) FeS centre, and e, f) heme b.See also Tables S12-S14.The MM and QM/MM optimisations were initiated from independent snapshots followed by a geometry optimisation.The MM system was minimised for 10000 steps, while the QM/MM system was minimised until energy convergence threshold of 0.006 kcal mol -

Table S2 .
List of subunits in the simulation models.

Table S5 .
Calculated average redox midpoint potentials of cofactors based on MD simulations (see TableS1for simulation number).All values are given in mV.

Table S7 .
15om types and partial charges for MQ cofactors calculated in different oxidation states.QM models included one isoprenoid unit and were optimized at B3LYP/def2-SVP level of theory (see Methods) while quinone tail charges were taken from CHARMM36m force field15.

Table S8 .
Atom types and partial charges for the FeS centre in different protonation and oxidation states relevant for the Rieske protein.Charges were calculated at B3LYP/def2-TZVP(Fe,S)/def2-SVP (C, H, N) using antiferromagnetic coupling (see Methods).

Table S10 .
Atom types and partial charges for heme c cofactor covalently bond to two Cys and ligated to one His and one Met residue in reduced (Fe +2 ) and oxidized state (Fe +3 ) calculated at B3LYP/def2-TZVP(Fe)/def2-SVP(N,C,O,H) (See Methods).

Table S11 .
Atom types and partial charges for Cu active site in SodC ligated to three His residues and a hydroxyl for the oxidized state (Cu II ) obtained at B3LYP/def2-TZVP(Cu)/def2-SVP (N, C, O, H) level of theory (See Methods).

Table S12 .
Comparison of MM and QM optimised quinone coordinates.Distances are given in Å and angles in degrees.See also Fig.S23and Methods.

Table S13 .
Comparison of MM and QM optimised coordinates of the FeS Rieske centre.Distances are given in Å and angles in degrees.Leading numbers before the atom name denote the ligand (2/3 = Cys, 4/5 = His).See also Supplementary Fig.S23and Methods.

Table S14 .
Comparison of MM and QM optimised coordinates of heme b.Distances are given in Å and angles in degrees.See also Supplementary Fig.S23and Methods.

Table S15 .
Calculated pKa values of selected residues in the Qo1a and Qi sites.The calculations were performed for Complex III (PDB ID: 6ADQ) 2 with bound quinone before (6adq) and after (6adq opt) geometry optimisation of sidechains.The table reports values for each protomer.